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Flow Cytometry01:23

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The development of flow cytometry techniques began in 1934 with initial attempts by Andrew Moldavan, a bacteriologist who counted the cells in a flowing capillary system. Moldavan pumped cells through a capillary tube focused under a microscope for visualization. The invention of photometry allowed the measurement of differentially-stained cells, and Louis Kamentsky developed the first multiparameter flow cytometer in 1965 to identify and count the cancer cells in cervical tissue specimens.
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Methods to Assess Microbial Populations01:30

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Assessing microbial populations is crucial for understanding microbial roles in health, ecology, and industry. Various complementary techniques—both culture-based and molecular—enable detailed analysis of microbial abundance, diversity, and function.Viable Plate CountThe viable plate count is a traditional culture-based method used to estimate the number of living microbes in a sample. After serial dilution, the sample is spread onto nutrient agar plates. Each viable cell forms a...
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Updated: Apr 12, 2026

Characterization of Aquatic Biofilms with Flow Cytometry
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A flow cytometric approach to quantify biofilms.

Monique Kerstens1, Gaëlle Boulet, Marian Van Kerckhoven

  • 1Laboratory of Microbiology, Parasitology and Hygiene (LMPH), S7, Faculty of Pharmaceutical, Biomedical and Veterinary Sciences, University of Antwerp, Universiteitsplein 1, 2610, Wilrijk, Belgium.

Folia Microbiologica
|May 8, 2015
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Summary

Flow cytometry (FCM) offers a rapid and precise method for quantifying viable cells within biofilms. This technique, using TO-PRO(®)-3 iodide, overcomes limitations of traditional methods for biofilm analysis.

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Concurrent Quantification of Cellular and Extracellular Components of Biofilms
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Area of Science:

  • Microbiology
  • Biotechnology
  • Analytical Chemistry

Background:

  • Biofilms are microbial communities crucial in clinical, industrial, and environmental settings.
  • Current methods for biofilm quantification are often time-consuming and do not enumerate viable cells.
  • Accurate enumeration of viable cells in biofilms is essential for effective control and understanding.

Purpose of the Study:

  • To introduce and validate flow cytometry (FCM) with TO-PRO(®)-3 iodide as a fast and precise method for viable biofilm cell quantification.
  • To optimize biofilm removal and dissociation techniques for single-cell suspension suitable for FCM.
  • To compare FCM quantification with traditional crystal violet staining for biofilm analysis.

Main Methods:

  • Developed and optimized a protocol for removing and dissociating mature biofilms (Candida albicans, Escherichia coli) into single-cell suspensions.
  • Utilized scraping, rinsing, and sonication for efficient biofilm dissociation.
  • Employed flow cytometry (FCM) with the viability dye TO-PRO(®)-3 iodide for cell enumeration.
  • Compared FCM results with crystal violet staining for biofilm biomass quantification.

Main Results:

  • A combination of scraping, rinsing, and sonication effectively produced single-cell suspensions from biofilms.
  • Flow cytometry (FCM) demonstrated good intraday precision (approximately 5%) for biofilm sample measurements.
  • FCM and crystal violet assays generated comparable biofilm growth curves for both E. coli and C. albicans.
  • FCM provided a rapid and precise quantification of viable cells within biofilms.

Conclusions:

  • Flow cytometry (FCM) using TO-PRO(®)-3 iodide is a validated, fast, and precise alternative for quantifying viable cells in biofilms.
  • This method overcomes the limitations of traditional techniques by enabling viable cell enumeration.
  • The optimized protocol facilitates accurate FCM analysis of microbial biofilms.